Antibacterial efficacy of Moringa oleifera leaf extract against pyogenic bacteria isolated from a dromedary camel (Camelus dromedarius) abscess

Background: Abscess in camel is one of the most important bacterial infections. It causes anemia and emaciation, resulting in an economic loss due to carcass condemnation and a decrease in reproductive and production efficiency. Aim: This investigation aimed to isolate the bacteria from abscesses in camels and evaluate the antibacterial activity of Moringa oleifera extracts. Materials and Methods: Disk diffusion method and minimum inhibitory concentration were used for the evaluation of the antibacterial activity of M. oleifera extracts against isolated bacteria from camel abscesses. Results: The isolated bacteria were displayed relatively as follows: Corynebacterium pseudotuberculosis (30.4%), Staphylococcus aureus (25.8%), Escherichia coli (17.8%), Corynebacterium ulcerans (10.5%), Klebsiella pneumoniae (8.5%), Pseudomonas aeruginosa (8.5%), Micrococcus spp. (6.7%), Proteus vulgaris (5.2%), Citrobacter spp. (4.2%), and Staphylococcus epidermidis (1.7%). The drugs of choice for Corynebacterium isolates were ciprofloxacin and trimethoprim/sulfamethoxazole, whereas amikacin, ciprofloxacin, gentamicin, neomycin, novobiocin, streptomycin, and vancomycin were for Staphylococcus isolates. Moreover, the ethanol extracts of M. oleifera showed higher antibacterial efficacy than the cold aqueous extracts. Conclusion: M. oleifera is considered one of the new infection-fighting strategies in controlling pyogenic bacteria responsible for camel abscesses.


Introduction
In the past, researchers paid only a little attention to camel research. However, numerous bacterial, viral, and parasitic diseases have been recorded. Camel abscesses have received little attention with the bulk of research directed toward historic diseases of the camel. In addition, in abscesses, there are both economic and public health hazards, which generally result in economic losses due to the condemnation of infected carcasses or part of it, leading to nutritional problems [1]. Camels' breeding could be done to overcome Egyptian's needs for animal protein [2]. Certain breeds of camel can live in more humid environments; the major breed is the dromedary of the Nile Delta of Egypt [3].
There are few literatures on camel research, consequently, no recent data related to economic losses due to abscesses. However, abscesses in animals cause tremendous economic losses such as decreased production of animal meat, skin, and wool. Animals with abscess become anemic and emaciated, due to toxins produced from it, resulting in loss of the animal's value and decrease in its reproductive and productive efficiency [4][5][6]. Infected animals had a poor physical condition, decreased fertility and condemnation of carcasses totally or partially at abattoirs [1,7,8].
Camel infections due to pyogenic bacteria such as Corynebacterium pseudotuberculosis, Corynebacterium pyogenes, Streptococci spp., Staphylococci spp., Corynebacterium ulcerans, Rhodococcus, Escherichia coli, Citrobacter spp., Klebsiella pneumoniae, Proteus spp., and Pseudomonas aeruginosa have been commonly reported in many areas [8][9][10]. In dromedaries, cervical and sciatic lymph nodes were mainly affected. Lymphangitis and suppurative lymphadenitis have been recorded in camel abscesses [11,12]. Camel abscess results in the entrance of the causative organism through the damaged skin and mucous membrane ended by reaching the regional lymph node then causes inflammatory and necrotic changes [13].
Available at www.veterinaryworld.org/Vol.12/June-2019/12.pdf Moringa species is used in the medicinal field worldwide due to its pharmacological activities and considerable medicinal compounds. The most common species of Moringa genus is Moringa oleifera which has rich sources of various phytochemical compounds including glucosinolates and has antibacterial activity [14]. According to the World Health Organization, 80% of the populations in developing countries prefer to use herbal extract and their active components as traditional medicine therapy [4]. M. oleifera is commonly known as drumstick and has many active components such as alkaloids, tannins, flavonoids, saponins, and triterpenoids [15,16] with potent anthelmintic activity [17] and antibacterial effect [18]. The phytochemical analysis of M. oleifera showed its bioactive compounds [19,20] with their pharmacological activity [21]. M. oleifera has a broad safety margin for human and animal consumption [22]. Disk diffusion method was used for the evaluation of the antibacterial activity of Moringa extracts, and the significant difference of inhibition zones appeared. P. aeruginosa, Enterococcus faecalis, and Staphylococcus aureus were used to assess the antibacterial effect of the extract [18,23].
This investigation aimed to isolate the bacteria from abscesses in camels and evaluate the antibacterial activity of M. oleifera extracts.

Ethical approval
The study was performed according to the Guide for the care and use of Laboratory animals and Ethical Approval of Animal Rights according to Committee, National Research Centre, Egypt.

Sampling
One hundred and seventy pus swabs were collected from 30 living and 70 slaughtered male camels (7-9 years old) that were obtained from El-Monib abattoir, Giza. These samples were obtained from external abscesses involving superficial lymph nodes and subcutaneous tissue, especially at the head, neck, and shoulder regions. Internal abscesses were mainly seen in the lungs, liver, and bronchial lymph nodes. According to Hatem et al. [5], the swabs placed in ice bags were transported through Cary-Blair (Difco) transport medium to the Department of Microbiology and Immunology, Veterinary Division, National Research Centre (NRC).
The collected swab samples were streaked onto blood agar plates (nutrient agar 13 g/L containing 5% citrated sheep blood), MacConkey agar plates (52 g/L), and mannitol salt agar 7.5% plates (111 g/L). All samples were streaked in duplicate plates and were incubated aerobically and anaerobically at 37°C for 24 h and 37°C for 48-72 h, respectively.

Identification of bacterial isolates
Bacterial isolates were microscopically identified according to Cruickshank et al. [24]. Biochemical identification of Gram-positive coccobacilli was carried out according to Funk et al. [25]. Biochemical identification of Gram-positive cocci and Gram-negative bacteria was carried out according to Quinn et al. [26] and Cruickshank et al., respectively [24].

Antibiotic sensitivity test for identified strains
Fifteen standard antibiotic disks were used against the isolated bacteria after preparation of the standardized bacterial inoculums matching with 0.5 McFarland tubes (10 8 colony-forming unit [CFU]/ml). Then, 25 µl of the inoculum was distributed on Muller-Hinton agar plates and incubated at 37°C for 24 h. The degree of sensitivity was determined by measuring the inhibition zone. The result was interpreted according to Dzotam et al. [16].

Plant material of M. oleifera
Moringa leaf powder was obtained from Moringa Unit, NRC. It was collected from a farm of the NRC in Al-Nubaria which is situated along Cairo/ Alexandria desert road. The plant material was collected in January 2018 and presented in the study.

Cold aqueous extracts of M. oleifera leaves
100 g of M. oleifera leaf powder was weighed out and dissolved in 400 ml of cold distilled water into a conical flask stoppered with rubber corks and left for 7 days with occasional shaking (10 times/day). The mixture was filtered off using a sterile filter paper (Whatman no. 1) into a clean conical flask and subjected to water bath evaporation where the aqueous solvent was evaporated at its boiling temperature of 100°C. The standard extracts obtained were then stored in a refrigerator at 4°C for antibacterial activity test [27].

Hot aqueous extracts of M. oleifera leaves
The same protocol as in cold water treatment was used with 30 min of boiling while the plant material was dipped in distilled water.

Ethanol (95%) extracts of M. oleifera leaves
Here, the same procedure as in cold water treatment was followed.

Test microorganisms
All the isolated bacteria from camel abscesses were used to assess the antibacterial effect of M. oleifera. It included C. pseudotuberculosis, C. ulcerans, and S. aureus as Gram-positive bacteria and E. coli, K. pneumoniae, Citrobacter spp., Proteus vulgaris, and P. aeruginosa as Gram-negative bacteria.

Antibacterial assay of M. oleifera
The antibacterial activity of the three different samples, namely, (1) cold water extract (CWE) of leaves, (2) hot water extract of leaves, and (3) ethanol extracts (EEs) of leaves, was individually tested against the studied bacteria. In vitro antibacterial test was then carried out by disk diffusion method [28,29] using 25 µl of the standardized bacterial suspension of the tested bacteria (10 8 CFU/ml) spread on plates. The disks (6 mm in diameter) were impregnated for different samples with 10 µl of 0.1 g/ml (100 mg/disk), followed by air drying, and were placed on seeded agar plates. Negative controls were prepared using the same solvents to dissolve the plant extracts. Tetracycline (TE) (30 µg/disk) was used as a positive control to determine the sensitivity of bacterial strain. The plates were incubated at 37°C for 24 h. The antimicrobial activity was evaluated by measuring the zones of inhibition against the tested bacteria.

Minimum inhibitory concentration (MIC)
The MIC of different samples of M. oleifera was determined by two-fold serial dilution method [14]. Serial dilution of 100 mg/ml for rest of the samples were separately done to achieve 50, 25, 12.50, 6.25, 3.12, 1.56, 0.78 mg/ml and 390, 195, 97 µg/ml concentration were used for MIC determination. Briefly, 100 µl of varying concentrations of samples were added into the test tubes separately, containing 9 ml of the standardized suspension of tested bacteria (10 8 CFU/ml). The test tubes were incubated at 37°C for 24 h. Controls were used with the test organisms, using distilled water instead of the plant extract. The least concentration of the samples with no visible growth was taken as the MIC [30].

Statistical analysis
To evaluate associations between variables (antibiotic profiles), the data were analyzed statistically using Student's "t-" test, showing mean + standard deviation [31].
Corynebacterium spp. colonies were appeared smooth, white in color, opaque, flat, circular, and small in size. Moreover, they had a narrow zone of ß-hemolysis on blood agar. The biochemical characteristics of C. pseudotuberculosis were positive catalase, urease, glucose, maltose fermentation and negative starch and trehalose fermentation, negative gelatin liquefaction, and nitrate reduction. C. ulcerans exhibited positive catalase, urease, gelatin liquefaction and fermented glucose, maltose, starch, and trehalose, with negative nitrate reduction test. On the other hand, Gram-positive cocci on nutrient agar was golden yellow in color; was smooth, opaque, circular, and medium in size; and was surrounded by the zone of ß-hemolysis on blood agar but was yellow in color on mannitol salt agar. While the non-hemolytic white colonies on blood agar exhibited pink colonies on mannitol salt agar. S. aureus was biochemically identified by catalase and coagulase positive, fermented maltose, trehalose, mannitol, and sucrose. While, S. epidermidis was coagulase negative, catalase positive and fermented sucrose only. Micrococcus isolates showed catalase positivity, coagulase negativity, and oxidative reaction (O) in the O/F test. Moreover, the Gram-negative bacilli were smooth, flat, circular and medium-sized colonies; either lactose fermenter colonies (appeared pink on MacConkey agar) or non-lactose fermenter (colorless). The biochemical characteristics of the isolated Gram-negative bacteria revealed the isolation of E. coli (17.8%), P. aeruginosa (8.5%), K. pneumoniae (8.5%), P. vulgaris (5.2%), and Citrobacter spp. (4.2%). The antibiotic sensitivity test is shown in Table-1.

Antibacterial activity of M. oleifera extracts
The antibacterial activity of cold water, hot water, and EE of M. oleifera is shown in Table-2. The EE of the leaves displayed a pronounceable better antibacterial effect against all the tested C. pseudotuberculosis, C. ulcerans, S. aureus, E. coli, K. pneumoniae, Citrobacter

Discussion
M. oleifera is considered one of the new infection-fighting strategies in controlling pyogenic bacteria responsible for camel abscesses. Improvement of camels' health and breed should be kept in mind for the economic significance of camel meat and milk. [2]. The appearance of abscesses in camel creates a marketing problem due to the decline of the meat quality and quantity and condemnation of the affected portions and internal organs. Isolation of Corynebacterium species, S. aureus, Streptococci, Rhodococcus, E. coli, K. pneumoniae, Citrobacter spp., P. vulgaris, and P. aeruginosa from lymphadenitis and abscesses in camels had been reported in the literature [1,[8][9][10][11][12].
The present work focuses on the isolation and identification of bacteria causing abscess in camel. The results of the survey on the prevalence of abscesses in camels resulted in the isolation of C. pseudotuberculosis (30.4%), S. aureus (25.8%), E. coli (17.8%), C. ulcerans (10.5%), P. aeruginosa (8.5%), K. pneumoniae (8.5%), Micrococcus spp. (6.7%), P. vulgaris (5.2%), Citrobacter spp. (4.2%), and S. epidermidis (1.7%). C. pseudotuberculosis was the highest isolated one followed by S. aureus and E. coli. These organisms were previously reported as a cause of camel abscess by Wernery [6], Hassan et al. [7], Wernery [8], Berlin [11], and Wernery and Kinne [12]. The Gramnegative bacteria did not isolate alone in an abscess, but they were associated with Corynebacterium spp. and/or Staphylococcus spp. Further investigations are needed to know if they can induce abscess alone or they come as a secondary infection.
An antibiogram study was assessed on all isolates; Table-1 clearly indicates that ciprofloxacin (100%), trimethoprim/sulfamethoxazole (100%), streptomycin  [34], and Mohan et al. [35].  [42,43]. The present investigation illustrated that the disk diffusion method was used for the evaluation of the antibacterial activity of M. oleifera extracts. The EEs showed higher antibacterial effect than the cold aqueous extracts (Figure-1). Hence, the extraction process with an organic solvent such as ethanol provided a better antibacterial activity than the processes of soaking, obtaining decoction, and boiling of the plant in water, which agrees with the finding of Nair et al. [44]. In this investigation, the greatest inhibition zones were found in EE against all the bacteria tested, which were higher than that of antibiotic TE (30 µg/disk) (Figure-1). In earlier studies, researchers reported that plant extracts showed less antibacterial effect on bacteria [45,46]. The pharmacological activities and considerable medicinal compounds of M. oleifera have high antibacterial effect when compared to the broad-spectrum TE antibiotic [30,47]. Today, most bacteria are multidrug-resistant [5,[48][49][50][51]. Natural antioxidants such as ascorbic acid, flavonoids, phenolics, and carotenoids are obtained from Moringa leaves [52]. It was reported that they have novel active compounds which possess an antibacterial effect and can overcome the multidrug resistance problem [16,53]. Hence, M. oleifera is considered one of the new infection-fighting strategies in controlling multi-drug resistant pathogenic bacteria. As well as, it has a wide safety margin for human and animal consumption.

Conclusion
In the current study, it can be concluded that the most predominant pyogenic bacteria isolated from camel abscesses were C. pseudotuberculosis, S. aureus, and E. coli. An effective infection control program is needed with a highly effective antibacterial agent/s because antibiotic resistance is common in Egyptian isolates. The bactericidal effect of M. oleifera leaf extracts was determined against the isolated bacteria. It further discusses optimal conditions for the extraction of essential compounds responsible for the elimination of pathogenic bacteria.